The objectives of this project are to discover the enzymatic pathways for genetic recombination and to construct an in vitro system for recombination of DNA molecules. An ATP-dependent DNase from H. influenzae Rd is implicated in recombination and has been purified and characterized. It cleaves double-stranded linear DNA molecules into large fragments with long single-stranded ends, hydrolyzing large amounts of ATP to ADP during the process. We are investigating the mechanism of this reaction by looking for enzymatic or DNA intermediates and exchange reactions. We are visualizing the fragments by electron microscopy and hope to observe enzyme molecules bound to DNA. We are correlating ATP hydrolysis with the apparent "melting" of the DNA to produce the single-stranded ends. We are using electron microscopic observation to investigate reannealed fragments which look similar to some "recombinant"-like molecules observed in phage-infected systems. We are further investigating mutants deficient in the DNase activity.